ORIGINAL RESEARCH ARTICLE published: 16 September 2014 doi: 10.3389/fpls.2014.00481 Chemical and structural analysis of Eucalyptus globulus and E. camaldulensis leaf : a lipidized region

Paula Guzmán1*, Victoria Fernández1*, José Graça 2 , Vanessa Cabral 2 , Nour Kayali 3 , Mohamed Khayet 4 and Luis Gil 1 1 Forest Genetics and Ecophysiology Research Group, School of Forest Engineering, Technical University of Madrid, Madrid, Spain 2 Instituto Superior de Agronomia, Centro de Estudos Florestais, Universidade de Lisboa, Lisboa, Portugal 3 Mass Spectrometry Unit, Faculty of Chemistry, University Complutense of Madrid, Madrid, Spain 4 Department of Applied Physics I, Faculty of Physics, University Complutense of Madrid, Madrid, Spain

Edited by: The plant has traditionally been conceived as an independent hydrophobic layer Anja Geitmann, Université de that covers the external epidermal cell wall. Due to its complexity, the existing relationship Montréal, Canada between cuticle chemical composition and ultra-structure remains unclear to date. This Reviewed by: study aimed to examine the link between chemical composition and structure of isolated, Mary Lai Preuss, Webster University, USA adaxial leaf cuticles of Eucalyptus camaldulensis and E. globulus by the gradual extraction Heiner Goldbach, University of Bonn, and identification of lipid constituents (cutin and soluble lipids), coupled to spectroscopic Germany and microscopic analyses. The soluble compounds and cutin monomers identified could *Correspondence: not be assigned to a concrete internal cuticle ultra-structure. After cutin depolymerization, a Victoria Fernández and Paula Guzmán, cellulose network resembling the cell wall was observed, with different structural patterns Forest Genetics and Ecophysiology Research Group, School of Forest in the regions ascribed to the cuticle proper and cuticular layer, respectively. Our results Engineering, Technical University of suggest that the current cuticle model should be revised, stressing the presence and Madrid, Ciudad Universitaria s/n, major role of cell wall polysaccharides. It is concluded that the cuticle may be interpreted Madrid 28040, Spain e-mail: [email protected]; as a modified cell wall region which contains additional lipids. The major heterogeneity of [email protected] the plant cuticle makes it difficult to establish a direct link between cuticle chemistry and structure with the existing methodologies.

Keywords: cell wall, cuticle, cutin, eucalypt, leaf, lipids, polysaccharides, ultra-structure

INTRODUCTION alcohols, alkanes, aldehydes, esters and ketones) and aromatic The cuticle is an asymmetric, composite layer covering the sur- compounds (Jetter et al., 2006). Polysaccharides present in the face of most aerial organs of plants (Tyree et al., 1990; Fernández cuticle are similar to those found in the cell wall (i.e., cellulose, and Brown, 2013), which is mainly extruded into the external hemicelluloses and pectins; López-Casado et al., 2007; Guzmán wall of epidermal cells (Pollard et al., 2008; Javelle et al., 2010). et al., 2014b). Based on the relative abundance of chemical con- Due to its location at the interface between the plant and the stituents (Schmidt and Schönherr, 1982; Heredia, 2003; Samuels surrounding environment, the cuticle plays an array of crucial et al., 2008), the cuticle has been conceived as a hydrophobic protective and physiological functions (Remus-Emsermann et al., membrane. 2011; Serrano et al., 2014). Additionally, it has a role in organ The cuticle has been generally described as having three dif- growth and differentiation (Kutschera and Niklas, 2007; Takada ferent layers from the external to the internal surface, i.e.,: (i) and Iida, 2014). the epicuticular wax (EW) layer, (ii) the cuticle proper (CP), and The multi-functional character of the plant cuticle is pro- (iii) the cuticular layer (CL; von Mohl, 1847; Jeffree, 2006). It has vided by its complex and heterogeneous chemical and physical been traditionally assumed that cutin/cutan and waxes are found nature (Gouret et al., 1993; Guzmán et al., 2014a). From a chem- both in the CP and CL, while the presence of polysaccharides is ical viewpoint, the cuticle is generally defined as a cutin and/or restricted to the CL (von Mohl, 1847; Jeffree, 2006). By contrast, cutan polymer matrix, with epicuticular waxes deposited on to the presence of cellulose and pectins in both cuticular layers has its outer surface, embedded intracuticular waxes and phenolics, been recently reported (Guzmán et al., 2014b). For various species and polysaccharides stemming from the cell wall (Domínguez and organs, different patterns of cuticular ultra-structure have et al., 2011). Cutin is formed by a network of inter-esterified, been categorized on the basis of transmission electron microscopy hydroxy and hydroxy-epoxy C16 and/or C18 fatty acids (Kolat- (TEM) observations (Holloway, 1982; Jeffree, 2006). However, a tukudy, 1980), sometimes linked with glycerol (Graça et al., 2002). fair degree of cuticular structural variation may arise from e.g., The chemical composition of an alternative cuticle matrix poly- the TEM sample preparation procedure implemented as recently mer named cutan remains controversial (Tegelaar et al., 1991; shown by Guzmán et al. (2014a). Deshmukh et al., 2005). Cuticular waxes (soluble cuticular lipids) The cuticular chemical composition and/or ultra-structure of are complex mixtures of aliphatic (mainly long-chain fatty acids, a few organs and species has been studied by gradual extraction

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and analysis of different cuticular fractions (e.g., Fernández et al., temperature (23–25◦C) for 1 month, manually shaking the flasks 2011, 2014; Tsubaki et al., 2013; Belge et al., 2014), and using at frequent time intervals. After the extraction period, clean intact diverse microscopic means (e.g., Canet et al., 1996; Casado and adaxial cuticles were selected, thoroughly washed in deionized Heredia, 2001; Koch et al., 2004). The potential link between , air-dried, and stored for further use. chemistry and structure of different cuticular fractions or spe- cific molecular constituents has been examined chiefly focusing on EXTRACTION AND DETERMINATION OF SOLUBLE COMPOUNDS EW (e.g., Baker, 1982; Barthlott et al., 1998; Jeffree, 2006). Inte- Isolated cuticles were cut into small pieces, wrapped in filter grative studies on the internal cuticle are however, scarce (e.g., paper and successively extracted in 150 mL of dichloromethane Wattendorff and Holloway, 1982; Gouret et al., 1993; Viougeas (6 h; Sigma–Aldrich), ethanol (12 h; Sigma–Aldrich) and distilled et al., 1995; Yeats et al., 2012), and the existing relationship water (24 h) using a Soxhlet apparatus. Solvents were subsequently between chemical composition and structure remains unclear so evaporated to dryness with a rotary evaporator and the material far (Khayet and Fernández, 2012). In addition, there is currently a extracted was weighed and stored. Soxhlet extracted (SE) cuticles limited understanding of the potential cross-links and/or molecu- were also stored for further analysis. lar assemblies taking place between cuticular chemical constituents The cuticular material extracted in dichloromethane was ana- (Pollard et al., 2008; Kunst and Samuels, 2009). lyzed by gas chromatography-mass spectrometry (GC-MS). Sam- Given the chemical and physical complexity of the plant ples were filtered using polytetrafluoroethylene (PTFE) filters prior cuticle, there is a need for carrying out comprehensive investi- to injection in a GC-MS apparatus (GCVarian CP-3800 coupled to gations which may help us understand the cuticle in a holistic a MS ion trap Varian Saturn 2200, GC-ITMS, automatic injector manner. The high complexity of the cuticle together with an COMBI-PAL; CTC Analytics, Zwingen, Switzerland). The chro- array of technical constrains may lead to theoretical miscon- matographic conditions were as follows (86 min per run): injection ceptions concerning the structure, chemistry and function of volume 1 μL, with Helium as carrier gas (1.2 mL min−1), and the cuticle (Guzmán et al., 2014a,b). In this study we aimed at injector temperature 290◦C. The column (SLB-5 ms fused silica analyzing the link between ultra-structure and chemical com- capillary column, 30 m × 0.25 mm × 0.25 μm film thickness; position (focusing on lipids) of the leaf cuticle, following an Supelco, Sigma–Aldrich) was set to 40◦C (3 min), heating rate integrative approach. For this purpose Eucalyptus camaldulen- of 10◦C min−1 up to 290◦C (18 min), and 20◦C min−1 up to sis and E. globulus were used as model species due to their 310◦C (13 min). The MS conditions were: 70 eV ionization volt- major importance for forestry production (Guzmán et al., 2013). age, 150◦C trap temperature, 20–550 units of mass scan range Furthermore, despite such species are native to different habi- with 8 min solvent delay. The compounds were identified and tats their taxonomic proximity may involve a certain degree of quantified by comparing their mass spectra with NIST library cuticular similarity. By merging different analytical and electron spectra and analyzing the corresponding peaks of GC-MS ion microscopy techniques before and after the selective extraction chromatograms. and identification of chemical constituents, the following ques- tions were addressed: (i) is the adaxial leaf cuticle of both species EXTRACTION AND DETERMINATION OF CUTIN CONSTITUENTS structurally and chemically similar?, (ii) how does the gradual For the two species, 80 mg of dehydrated (oven-dried at 40◦C extraction of cuticular components affect cuticular ultra-structure before use), SE cuticles were used for cutin depolymerization. as compared to intact tissues?, and (iii) is it currently possible to Samples were refluxed in 5 mL of 0.1 M sodium methoxide solu- establish a relationship between cuticular chemical composition tion (Sigma–Aldrich) in methanol by heating in an oil bath at 80◦C and structure? for 3 h, and continuous stirring. For separation of de-esterified (fil- tered) and residual (non-filtered) material from the methanolysis MATERIALS AND METHODS process, mixtures were vacuum filtered using 1 μm pore PTFE fil- ◦ PLANT MATERIAL AND CUTICLE ISOLATION ters. The residual material was oven-dried under vacuum at 40 C The adaxial surface of fully expanded, mature, undamaged leaves overnight, weighted for the determination by mass-loss, of the of E. camaldulensis Dehn and E. globulus Labill was analyzed either total content of cuticle de-esterified material (one replicate) and intact or after enzymatic isolation of the cuticle. Leaves were col- saved for further examination. lected at late summer from the apex of external, west-facing shoots The total volume of de-esterified material was divided for glyc- of 30–35 years old trees growing at the Forest Engineering School erol and cutin monomer and precursor identification (1:4, v:v), Arboretum (Technical University of Madrid, Spain). For each and handled separately. For glycerol analysis, the de-esterified species, 6 and 30 leaves were selected for intact leaf and isolated aliquot was sampled as such. For cutin monomer identification, cuticle analyses, respectively. the respective aliquot was acidified to pH 6 with 0.05 M sulfuric Prior to cutting the central part of the leaf blades in small acid in methanol, and the solvent was subsequently removed in a pieces for cuticle isolation, leaf margins and midribs were removed rotary evaporator at 50◦C. The obtained residue was partitioned with a scalpel. Leaf cuticles were subsequently isolated in a in dichlorometane:water (1:1, v:v; 35 mL each) using a separat- solution containing 5% cellulase and 5% pectinase (Novozymes, ing funnel. The upper aqueous, clear phase was discarded and the Bagsvared, Denmark), plus 1% polyvinylpyrrolidone (Sigma– organic, opaque phase located below was partitioned again with Aldrich, Munich, Germany) and 2 mM sodium azide, which was the same volume of water. set to pH 5.0 by adding sodium citrate (Guzmán et al., 2014a). Tis- For GC-MS determination, aliquots (1 mL) of the de-esterified sues were maintained in solution (changed after 2 weeks) at room material and of the accumulated organic phases were collected.

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Aliquots were dried under a N2 flow and derivatized with N,O- and ImageJ 1.45s (National Institutes of Health, Bethesda, MD, bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosi- USA) softwares. Measurements were performed at periclinal areas, lane (BSTFA with 1% TMCS, Sigma–Aldrich) and pyridine at excluding zones located in the vicinity of cuticular pegs, and also a 1:1 (v:v) ratio. After 15 min staging in an oven at 60◦C, the EW layer. Micrographs corresponding to extremely thick or solutions were analyzed with an Agilent 5973 MSD GC-MS (Agi- thin cuticle sections or with no clear limits between adjacent lent, Santa Clara, CA, USA) in a DB-5 ms column (60 m, cuticular regions were discarded. Finally, 15 micrographs of each 0.25 mm i.e., 0.25 μm film thickness; Agilent), with the fol- sample (corresponding to three 4 mm2 cuticle pieces) were selected lowing GC conditions: injector 320◦C, initial oven temperature for thickness measurement, with 10–20 repetitions. 100◦C (5 min), heating rate of 8◦C min−1 up to 250◦C, and 2.5◦C min−1 up to 320◦C (20 min). The MS conditions were: FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY source 220◦C,quadrupole 150◦C,electron ionization energy 70 eV. Infrared spectra of cuticle samples (i.e., intact, SE cuticles, and Cutin monomers were identified by library search (NIST 98, SED residues), were obtained with an attenuated total reflectance Wiley 6), comparison to standards and/or mass spectra interpreta- (ATR) accessory (MIRacle ATR; PIKE Technologies, Madison, WI, tion (Graça et al., 2002). Monomer quantification was calculated USA) coupled to a FTIR spectrometer (Nicolet Nexus 670–870; from the areas of the corresponding peaks in the CG-MS ion Thermo Fisher Scientific, Waltham, MA, USA). Spectra of intact chromatograms. As internal standards for glycerol and cutin samples were recorded in transmission mode in the range 4000– − − monomers, we used 1,12-dodecanediol (0.05–0.3 mg; ≥98.5% 400 cm 1 with 4 cm 1 resolution and accumulating 64 scans, and GC, Sigma–Aldrich) and methyl 12-hydroxystearate (0.05–0.1 mg; analyzed with Omnic v4.1b (Thermo Fisher Scientific) software. ≥99.0% GC, Sigma–Aldrich), respectively. A response factor of Two replicates per sample were analyzed. 0.33 in relation to the internal standard (1,12-dodecanediol) was used for the quantitation of glycerol. The internal stan- RESULTS dards were diluted in methanol, homogenized in a warm bath, ELECTRON MICROSCOPY OBSERVATIONS and added to the de-esterified material before derivatization Intact leaves and cuticles (Graça and Lamosa, 2010). The adaxial surface of E. camaldulensis and E. globulus leaves and intact cuticles had a rough topography conferred by EW and epi- ELECTRON MICROSCOPY dermal structures such as papillae and stomata (Figures 1A–D). Intact leaves, isolated cuticles, SE cuticles, and the residues The most prominent EW structures were plates with different ori- obtained after de-esterification of SE cuticles (subsequently called entations for E. camaldulensis and tubes for E. globulus. Plates SED residues) were examined by TEM and scanning electron were observed to densely cover the whole leaf and cuticle surfaces microscopy (SEM). with the exception of the tip of some papillae (Figures 1A,C), Gold-sputtered adaxial leaf surfaces and both the inner and while wax tubes were principally found around papillae and outer cuticle and residue surfaces (three replicates per sam- stomata (Figures 1B,D). The isolated cuticle topography was ple) were observed with a Philips XL30 SEM (Eindhoven, The smoother compared to that of intact leaves, chiefly due to the Netherlands) microscope. loss or distortion of some EW during the cuticle isolation process For TEM observation, samples were cut into 4 mm2 pieces and (Figures 1C,D). The cuticle inner sides revealed the epidermal fixed in 2.5% glutaraldehyde-4% formaldehyde freshly prepared cell surface shape (i.e., the upper periclinal wall with papillae, from paraformaldehyde (both from electron microscopy sciences and the anticlinal flanges or cuticular pegs; Figures 1E,F), and (EMS), Hatfield, PA, USA) for6hat4◦C, rinsed in ice-cold phos- showed a granular pattern especially noticeable in E. globulus phate buffer, pH 7.2, four times within a period of 6 h and left (Figure 1F). overnight. Tissues were post-fixed in a 1:1 2% aqueous osmium The cuticles of E. globulus and E. camaldulensis were between tetroxide (TAAB Laboratories, Berkshire, UK) and 3% potassium 5.5 to 9.5 μm and 2.5 to 5.0 μm thick, respectively (Figure 2). ferrocyanide (Sigma–Aldrich) solution for 1.5 h. They were then The CP of E. camaldulensis (100–250 nm thick; Figure 3A) and washed with distilled water (x3), dehydrated in a graded series E. globulus (350–500 nm thick; Figure 3B) cuticles represented of 30, 50, 70, 80, 90, 95, and 100% acetone (x2, 15 min each between 5 and 6% of the total cuticle thickness. The cuticle of E. concentration) and embedded in acetone-Spurr’s resin (TAAB globulus was about two times thicker and had a two to three times Laboratories) solutions [3:1, 2 h; 1:1, 2 h; 1:3; 3 h (v:v)] and thicker CP than E. camaldulensis. The thickness of the EW layer in pure resin overnight at room temperature (25◦C). Tissues were was not considered since the TEM sample preparation procedure finally embedded in blocks which were incubated for 3 days at may significantly affect EW composition and structure. 70◦C. Ultra-thin sections of three leaf and cuticle pieces per treat- In contrast to the EW layer, the remaining cuticular ultra- ment were cut, mounted on nickel grids and post-stained with structure was not significantly altered by the isolation process as Reynolds lead citrate (EMS) for 5 min. They were subsequently derived from comparing intact cuticle TEM micrographs either observed with a Jeol 1010 TEM (Tokyo, Japan) operated at 80 kV as isolated tissues or when attached to the leaf. According to the and equipped with a CCD megaview camera. nomenclature suggested by Holloway (1982), the cuticle of both The thickness of entire cuticle transversal sections and of species had a faintly lamellate CP, with alternate electron-lucent the different cuticular regions was measured on TEM micro- and electron-dense lamellae (Figures 3A,B). While the CL of E. graphs of leaves, intact and SE cuticles, and SED residues using camaldulensis cuticle showed a gradual decrease of the reticulum siViewer (Olympus Soft Imaging Solutions, Münster, Germany) toward the CP, an amorphous region could be identified between

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FIGURE 1 | Scanning electron microscopy (SEM) micrographs of the EW structures of E. camaldulensis (C) and E. globulus cuticles (D). Inner adaxial leaf surface and intact, isolated cuticles of E. camaldulensis side and detail of the granules in the periclinal cuticle of E. and E. globulus. Surface and detail of EW structures of E. camaldulensis (E) and E. globulus cuticles (F). Bars in insert images: camaldulensis (A) and E. globulus leaves (B). Outer side and detail of 5 μm (A,B,D,F),10μm (C,E). the CP and the reticulate inner region in the cuticle of E. globulus. of the EW were solubilized, some plates remained in E. camaldu- The reticulate and amorphous CL areas close to the CP can be lensis SE cuticles (Figure 4A), and a cracked EW layer was observed respectively, observed in Figures 3A,B. in E. globulus SE cuticles (Figure 4B). Whilst no major structural changes were noticeable in the inner side of SE cuticles, the gran- Soxhlet extracted cuticles ules observed in intact cuticles were mostly removed, and cavities The topography of the outer surface of SE cuticles became with a similar size were identified in SE periclinal cuticle areas smoother compared to intact tissues (Figures 4A,B). While most (Figures 4C,D).

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FIGURE 2 |Transversal sections of the adaxial cuticle and outer cell wall of E. camaldulensis (A) and E. globulus (B) intact leaves. cm: cuticular membrane, cw: cell wall. Bars: 2 μm.

Patches of EW crystalline projections were also observed in diffuse regions had a thickness similar or sometimes slightly below cuticular cross-sections. For both species, the CP lamellae disap- that of the CP of intact cuticles, as also recorded for the amorphous peared after Soxhlet extraction. After this process an amorphous, outer region of SE cuticles. The cellulose lamellae of the inner electron-lucent band arose which had a thickness slightly below or region had a mainly parallel orientation to both the periclinal within the range of the intact CP. The upper limit of this band was (Figures 3E–H) and the anticlinal cell wall underneath (data not a relatively thin, discontinuous, electron-dense line, which may shown), which was especially noticeable in E. camaldulensis SED correspond to non-solubilized waxes (Figures 3C,D). The Soxhlet residues (Figures 3E,G). The electron-dense line located in the EW extraction process did not seem to modify the CL reticulum, and layer position seemed not to be affected by the de-esterification no remarkable structural or thickness changes in the rest of this process, especially in E. camaldulensis (Figures 3E,G). The cellu- region were observed (data not shown). The presence of gran- lose fibrils localized in the innermost cuticle areas of SED residues ules in the inner surface of the cuticle was also noticed in TEM appeared ripped to some extent (e.g., Figure 3F). micrographs (data not shown). Cuticle chemical composition De-esterified residues The relative proportion of cuticular chemical constituents The structure of SED residues was disrupted as observed in the obtained after successive extractions are shown in Table 1. Both inner side of such tissues by SEM (Figures 4E,F). The concavity eucalypt species followed a similar trend concerning the relative of the periclinal cuticle appeared to increase (e.g., Figure 4E), amount of chemical constituents. The de-esterified material repre- while the anticlinal cuticle seemed to be more fragile and was not sented the greatest percentage, accounting for approximately half distinguishable in some areas (Figures 4E,F). The upper side of of the total cuticle weight, followed by Soxhlet extracted, soluble SED residues had a similar appearance to that of SE cuticles (data compounds. The remaining SED residues (mainly polysaccha- not shown). rides; Figures 5E,F) constituted the smallest cuticular fraction. A considerable cuticle thickness decrease of approximately 60– The dichloromethane-soluble extractives included aromatic 75% and 70–80% for E. camaldulensis and E. globulus cuticles was and chiefly aliphatic compounds. Aliphatic molecules accounted observed as a result of the de-esterification process (Figures 2 vs. for approximately 96% and 76% of the total amount of iden- 3E,F). This reduction largely occurred in association with the inner tified compounds for E. camaldulensis and E. globulus cuticles, cuticle part (i.e., the zones which were closer to the epidermal cell respectively. The relative amount of aromatics was hence six walls). This was evidenced by the longer cuticular pegs observed in times greater in E. globulus than in E. camaldulensis. Cyclic com- SED residues as compared to those of intact and SE cuticles (data pounds (both alicyclics and aromatics) constituted about 39% not shown) and by the presence of EW (e.g., Figures 3E,G). (E. camaldulensis) and 76% (E. globulus). Among E. camaldulen- The cell wall architecture was observed in the transversal sis extractives, the most abundant soluble compound types were sections of SED residues (Figures 3E–H). While diffuse cellulose aliphatic esters (26%), followed by primary alcohols (20%). For fibrils were detected in the outermost cuticle region, a lamel- E. globulus, terpenes and terpenoids represented the highest per- late, helicoidal cellulose pattern was noticed in the adjacent inner centage of extractives (45%, chiefly sesqui- and tri-terpenoids), region. The position within the cuticle of these regions corre- followed by phenolics (21%). Individually, [(Z)-octadec-9- sponded to that of the CP and CL (Figures 3G,H). The outer, enyl] (Z)-octadec-8-enoate (22%) and hentetracontan-1-ol (16%)

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α,ω-diacids were also identified and amounted to 17% (E. camal- dulensis) and 8% (E. globulus). For the two species, the number and total amount of C18 cutin monomers were higher than those of C16 monomers, with C18/C16 ratios of 1.6 (E. camaldulensis) and 3.1 (E. globulus). Hydroxylated monomers accounted for 76 and 72% of the total amount of compounds for E. camaldulensis and E. globulus samples. Among them, those having two hydroxyl groups were the most abundant (42%) in E. camaldulensis, fol- lowed by the identified three-hydroxyl-compound (22%, Table 2). By contrast, monomers having one hydroxyl group accounted for the greatest percentage (30%) in E. globulus, closely followed by those having two hydroxyl groups (28%). On the other hand, epoxy monomers constituted about 13 and 35% for E. camal- dulensis and E. globulus de-esterified material, respectively. The amount of hexadecanoic acid was relatively low as well as that of coumaric acid, the latter being only identified in E. camal- dulensis. Although representing a minor contribution, glycerol was present in both samples and its amount was twofold for E. camaldulensis.

FTIR spectroscopy The ATR-FTIR spectra of intact and SE cuticles, and of SED residues are shown in Figure 5. The spectra of E. camaldulensis and E. globulus intact cuticles (Figures 5A,B) indicate the presence of long-chain linear aliphatic compounds which were associated with the intense and thin bands at 2916 and 2917 cm−1 and at 2849 cm−1 [asymmetric and symmetric vibrations of the methy- lene groups, CH2; νa(CH2) and νs(CH2), respectively], as well as −1 −1 smaller bands at 1462 and 1463 cm and at 719 cm [CH2 scissoring and rocking vibrations; δscis(CH2) and δrock(CH2), respectively]. By comparing the intensities of these bands with those corresponding to the ester groups in the spectra of intact and chiefly SE cuticles (Figures 5C,D), it could be reckoned that these aliphatic compounds are chiefly ascribed to soluble lipids. The absorptions at 1713 and 1719 cm−1 and their shoul- −1 FIGURE 3 |Transmission electron microscopy (TEM) micrographs of the ders at 1686 and 1687 cm [C = O stretching vibration of adaxial leaf cuticles of E. camaldulensis and E. globulus both intact the carbonyl bonds; ν(C = O)] for E. camaldulensis and E. and after successive extractions. Outer regions of E. camaldulensis (A) globulus may correspond to soluble compounds with ketone or and E. globulus (B) intact cuticles. Outer regions of E. camaldulensis (C) and E. globulus (D) SE cuticles. Complete transversal section and detail of aldehyde functional groups which were identified in both sam- − the outer region of E. camaldulensis (E,G) and E. globulus (F,H) SED ples. These vibrations together with the vibrations at 1168 cm 1 residue. SE: Soxhlet extracted, SED: Soxhlet extracted, and depolymerized. and at 1105 and 1104 cm−1 [asymmetric and symmetric C–O–C CL: cuticular layer; CP: cuticle proper; cm: cuticular membrane; EW: ν ν epicuticular wax layer. Bars: 200 nm (A,B,G,H),500nm(C–F). The arrow stretching vibrations of the ester bonds; a(C–O–C)est and s(C– indicates an area of ripped cellulose fibrils as an example (F). O–C)est, respectively] may also be associated with esterified soluble lipid compounds and especially with the cutin in the polymer form. were the most abundant soluble lipids identified in E. camal- The low intensity bands at 1517 and 1518 cm−1 were related to dulensis cuticle. (1aR,4R,4aR,7R,7aS,7bS)-1,1,4,7-tetramethyl- the stretching of aromatic rings [ν(C–C) conjugated with (C = C) 2,3,4a,5,6,7,7a,7b-octahydro-1aH-cyclopropa[e]azulen-4-ol (16%) or ν(C-C)/(C = C)]. The middle intensity bands at 1030 and and 5-methoxy-2,2-dimethyl-10-(3-methylbut-2-enyl)pyrano[3,2- 1035 cm−1 were assigned to glycosidic bonds typical of polysac- g]chromen-8-one (13%) constituted the highest percentages of charides [ν(C–O–C)gly ]. The broad bands appearing at 3388 and soluble extractives for E. globulus cuticle. 3345 cm−1 [H-bonded, O–H stretching vibration; ν(O-H···O)] For the two species, the lipidic, de-esterified material (Table 2) were assigned to hydroxyl functional groups and could be related was found to be chiefly composed of aliphatic ω-hydroxyacids, to hydration. The assignment of other minor bands can be which represented approximately 62% (E. camaldulensis) and 69% found in Heredia-Guerrero et al. (2014). (E. globulus) of the total amount of such material (also including The spectra of SE cuticles (Figures 5C,D) showed an incre- unidentified long-chain aliphatic compounds). In both samples ment in the bands associated with ester functional groups. By

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FIGURE 4 | SEM micrographs of the adaxial leaf cuticles of E. E. camaldulensis (E) and E. globulus (F) SED residues. SE: Soxhlet extracted, camaldulensis and E. globulus after successive extractions. Outer side of SED: Soxhlet extracted, and depolymerized. Bars in insert images: 5 μm E. camaldulensis (A) and E. globulus (B) SE cuticles. Inner side and detail of (C,D,F),2μm (E). Arrows indicate areas were the anticlinal cuticle was not E. camaldulensis (C) and E. globulus (D) SE cuticles. Inner side and detail of visible (E,F). comparing the intensity of the bands corresponding to the asym- to hydrated polysaccharides. On the other hand, the spectra metric and symmetric vibrations of CH2 and that of the ester C–O of E. globulus SED residue still showed the presence of a rel- stretching vibration, it can be concluded that most of the CH2 can atively minor amount of non-solubilized long-chain aliphatic be assigned to cutin. The higher intensity of the bands associ- compounds (Figure 5F), which constitute the most noticeable ated with aromatic compounds in SE cuticles spectra as compared difference between the two species as derived from all the spectra to those observed in the spectra of intact cuticles indicated the analyzed. presence of aromatic compounds related to insoluble lipids and phenolics. DISCUSSION Finally, the spectra of SED residues (Figures 5E,F) exhibited In this study, we analyzed the chemical composition of the adax- an increase of the bands assigned to polysaccharides. In addition, ial leaf cuticle of E. camaldulensis and E. globulus in relation to the broad bands corresponding to hydroxyl groups increased with their ultra-structure, by merging different analytical and micro- respect to the intact and SE cuticle spectra, and may correspond scopic methods. Such species were selected as model due to their

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Table 1 | Chemical composition of adaxial cuticles of Eucalyptus taken into account only in few investigations (e.g., Matas et al., camaldulensis and E. globulus leaves after successive extractions 2004; López-Casado et al., 2007; España et al., 2014; Guzmán et al., expressed as weight percentages of the intact cuticles. 2014b).

Sample Soxhlet SE cuticle CUTICLE CHEMICAL COMPOSITION extractives (%%%) De-esterified Residual material According to the assignment of cuticular fractions after successive material (%%%) (SED residue; %%) chemical extractions, cutin (directly associated with de-esterified material; e.g., España et al., 2014) was the most abundant com- E. camaldulensis 38.3 48.2 13.5 ponent for the two eucalypt samples analyzed, accounting for E. globulus 37.3 53.6 9.1 48–54% of the total cuticle weight, followed by soluble lipids (organic solvent extractives; about 38–37%). Polysaccharides SE, Soxhlet extracted; SED, Soxhlet extracted and depolymerized. actually represented the lowest cuticle fraction (the residual material; 14.9%). commercial significance for forestry production. Since they belong Nevertheless, the different cuticle extraction processes may to the same genus, such leaf cuticles may additionally have chem- lead to significant chemical losses and to a potentially inaccurate ical and structural similarities which may however, be affected assignment of chemical groups. For example, it is likely that the by their different native habitat. Only few studies analyzed plant polysaccharide fraction is currently underestimated, in contrast cuticles following an integrative approach, and the link between to the possibly overestimated cutin fraction. The de-esterification cuticle chemistry and structure remains unclear. On the other process may have released additional chemical constituents which hand, the presence and role of cuticular polysaccharides has been are linked by ester bonds (e.g., waxes, phenolics, pectins, or

FIGURE 5 | ATR-FTIR spectra of the adaxial leaf cuticles of E. Soxhlet extracted and depolymerized. ν: stretching, δ: bending, a: camaldulensis (A,C,E) and E. globulus (B,D,F). Intact cuticles (A,B),SE asymmetric, s: symmetric, scis: scissoring, rock: rocking, est: ester, gly: cuticles (C,D), and SED residues (E,F). SE: Soxhlet extracted, SED: glycosidic bond.

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Table 2 | Cutin composition of isolated adaxial cuticles of noticeable in terms of different TEM cuticular ultra-structures. E. camaldulensis and E. globulus leaves. E. camaldulensis had a higher proportion of C18 chain-length monomers as compared to E. globulus (C18/C16 ratio of 3.1 Relative abundance (%%%) vs. 1.6, respectively). Additionally, E. camaldulensis had higher occurrence of monomers with mid-chain hydroxyl groups and Cutin components E. camaldulensis E. globulus a higher relative content of α,ω-diacids (17.0% vs. 8.1%) and glycerol (4.4% vs. 2.4%). The cuticle composition differences 16-Hydrohexadecanoic acid 2.4 1.3 recorded may lead to structural differences concerning e.g., the 9(7–10),16-Dihydroxyhexadecanoic 28.1 17.6 degree of reticulation or molecular arrangement, also consider- acid ing the occurrence of higher glycerol and α,ω-diacids amounts 9(10),18-Dihydroxyoctadecenoic acid1 0.0 5.1 in E. camaldulensis, as observed in suberins (Graça et al., 2002; 9-Epoxy-18-hydroxyoctadecanoic acid 9.5 28.6 Graça and Lamosa, 2010). 9-Epoxy-octadecane-1,18-dioic acid 3.0 4.8 9,10-Dihydroxyoctadecane-1,18-dioic 14.0 3.3 INTACT CUTICLE ULTRA-STRUCTURE acid The ultra-structure of E. camaldulensis and E. globulus cuticles is Epoxy-9(10),18-dihydroxyoctadecanoic 0.0 1.7 characterized by having an EW layer with different crystalline pro- jections (plates and tubes, respectively), a faintly lamellate CP and acid2 a CL with variable morphology. A fully reticulate CL was observed 9,10,18-Trihydroxyoctadecanoic acid 22.0 14.4 in E. camaldulensis cuticle, whereas an external amorphous CL was Coumaric acid 0.6 0.0 found between the CP and the reticulate internal CL of E. globulus. Hexadecanoic acid 0.4 0.2 Intact cuticle inner surfaces were additionally covered with granu- Glycerol 4.4 2.4 lar structures. The occurrence of a bi-layered CL, as observed for E. Unidentified3 15.6 20.6 globulus, has been shown in other young and mature leaf cuticles of different species (e.g., Schmidt and Schönherr, 1982; Watten- 1Position of double bond not assigned dorff and Holloway, 1982; Jeffree, 2006). The presence of granules 2Position of epoxide not assigned in the inner surface of isolated leaf cuticles has been previously 3Several long-chain aliphatic compounds reported (Oladele, 1983; Carpenter et al., 2007). In this study, we found inter-specific differences in relation to hemicelluloses), trapped in or strongly bound to cutin and/or the thickness, ultra-structure, and chiefly chemical composition polysaccharide networks. The constituents located in the inner of the two eucalypt cuticles analyzed. Such cuticle variations may cuticle regions were especially affected, as concluded from the be associated with genetic traits having an adaptive function in estimated thickness loss and occurrence of ripped cellulose fib- response to their native environment (leaves of a similar age were rils in SED residues. Furthermore, significant amounts of pectins, collected from E. camaldulensis and E. globulus trees grown under hemicelluloses, mineral elements and even cellulose may be lost similar environmental conditions). Cuticular differences between when keeping tissues in aqueous solution, e.g., during the cuti- species belonging the same genus have been previously reported cle isolation, Soxhlet extraction, or de-esterification processes. A with especial focus on EW (e.g., Hallam and Chambers, 1970; number of factors associated with e.g., the cuticle isolation pro- Riedel et al., 2007). cess, solubility of different lipid types in organic solvents, and/or physical constraints that may limit the access of the solvents and solutes employed for lipid extraction and cutin depolymerization, ASSESSING THE LINK BETWEEN CUTICULAR CHEMISTRY can ultimately influence the gross amount, quality and structure AND ULTRA-STRUCTURE of cuticular components. SOXHLET EXTRACTED CUTICLES Some of the Soxhlet extracted compounds identified in this The lamellate CP regions observed in intact leaf cuticles were study have been previously reported for E. camaldulensis and E. replaced by amorphous, electron-lucent bands after Soxhlet globulus (e.g., Li et al., 1997; Silvestre et al., 1997; Jeffree, 2006; extraction. These bands had a thickness in the same range or Steinbauer et al., 2009; Verdeguer et al., 2009). Differences in slightly below that of the corresponding intact CP. By con- relation to the occurrence and relative content of molecular con- trast, no remarkable changes were noticed between the CL of stituents and soluble compound types were observed between the intact and SE cuticles. X-ray diffraction and TEM studies per- two species. Aliphatic esters (26%) and terpenes and terpenoids formed on Hedera helix leaf cuticles both intact and after solvent (45%) were the most abundant compounds in E. camaldulensis extraction revealed similar results, suggesting an intermolecular and E. globulus cuticles, respectively. The amount of cyclic com- disorganization of the cuticles, particularly in the CP (Viougeas pounds was almost twofold higher for E. globulus as compared to et al., 1995). The appearance of the CL was also preserved E. camaldulensis (76 vs. 39%), with aromatics being six times more (Viougeas et al., 1995), alike in Agave americana extracted cuticles abundant in E. globulus (24 vs. 4%). (Wattendorff and Holloway, 1982). The CP thickness variation The dissimilar cutin monomer composition of the two eucalypt may be due to several phenomena which may also affect the CL species analyzed may lead to different cutin molecular archi- region, such as the loss of cuticular material and structure, or the tectures (Graça and Lamosa, 2010). This was however, not potential shrinkage of the tissues as affected by Soxhlet extraction

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or TEM sample preparation procedures. The influence of cuti- A significant amount of material was removed largely from cle position within one or between different leaves should also be the inner cuticle region, which experienced a thickness reduc- considered (Riederer and Schönherr, 1988). tion of approximately 60–80%. Working with A. americana leaf No clear relationship between soluble lipids composition and cuticles, Wattendorff and Holloway (1982) reported a thick- cuticle internal morphology could be established. As an excep- ness reduction of 40% or more from the inner side after cutin tion, E. camaldulensis epicuticular plates may correspond to depolymerization. Apart from the lower content and/or degree hentetracontan-1-ol (Jeffree, 2006). The chemical nature of the of polymerization of cutin located in the more exposed internal CP lamellae has been attempted to be identified in a few inves- CL, which may yield this region more susceptible to degradation tigations (see Jeffree, 2006), but remains unclear so far. Our (Wattendorff and Holloway, 1982), the role of further chemical results indicate that the electron-lucent lamellae of E. camal- compounds should also be considered as discussed above. dulensis and E. globulus CP are partially composed of soluble lipids as suggested by Domínguez and Heredia (1999) for the IMPLICATIONS OF THE RESULTS OBTAINED Clivia miniata leaf cuticle, but the presence of other free or The chemical and structural analyses performed with E. globulus chemically bound compounds cannot be discarded. The gran- and E. camaldulensis leaves, indicate that the cuticle of such species ular structures covering the inner, intact cuticle surface which may be interpreted as a lipidized cell wall region. This result is were removed during Soxhlet extraction may be associated in agreement with the viewpoint of Yeats and Rose (2013),who with soluble intracuticular lipids. The remaining granules may suggested that the cuticle can be considered a specialized lipidic correspond to insoluble lipids and/or cutin (i.e., cutinsomes; modification of the cell wall. Our results show that a polysaccha- Heredia-Guerrero et al., 2008). ride network may provide the basis for cuticle structure, a role which has been frequently assigned almost exclusively to cutin DE-ESTERIFIED RESIDUES (Kolattukudy, 1970). Cuticular lipid constituents may impregnate The de-esterification step led to substantial structural changes as and be intruded into such cell wall matrix, with waxes being also compared to the Soxhlet extraction process. While FTIR spectra deposited on to the outer cuticle surface, early at epidermal cell confirmed the major polysaccharide nature of the SED residues ontogeny (Jeffree, 2006; Guzmán et al., 2014b). The occurrence of (Heredia-Guerrero et al., 2014), TEM micrographs enabled us a polysaccharide network throughout the entire cuticle, with the to observe the cellulose architecture of the external cell wall, exception of the EW layer, may have significant bio-mechanical with a non-ordered, outer region and a larger multi-layered, implications and also contribute to the multi-directional transport helicoidal inner region (also visible in cuticular pegs). The rel- of water and solutes as suggested by Guzmán et al. (2014a,b). ative position within the cuticles and measurement of TEM It is likely that the actual proportion of polysaccharides is seri- micrographs of intact, SE cuticles and such SED residues indi- ously underestimated by current methods of cuticular handling cate that these regions may be ascribed to the CP and the CL, and analysis. The importance of polysaccharides as integral, essen- respectively. tial cuticle materials should be recognized for the development of One or the two cellulose fibril orientation patterns found in methodologies which allow a proper interpretation of the plant the SED residues have been previously reported for the epider- cuticle in biological, chemical, and physical terms. In light of the mal cell wall of a few species and organs (e.g., Roland et al., 1982; present study, the prevailing model which conceives the cuticle as Kutschera, 2008), and also for the H. helix leaf cuticle (Canet et al., a lipidic and hydrophobic extra-cellular layer should be reconsid- 1996). Thereby, the structure of the cellulose network seems to ered, highlighting its cell wall nature and heterogeneous character leave an imprint on the morphology of cuticular cross-sections in terms of polarity/apolarity and hydrophobicity/hydrophilicity. and may contribute to the different ultra-structure of the CP However, further studies with different plant organs, organ parts, (possibly with randomly oriented cellulose) and CL (with ordered and species will be required for proposing a broader plant cuticle cellulose). However, it is also possible that the removal of cuticu- model, and also for establishing a link between cuticle chemistry, lar material may have disrupted the orientation and arrangement structure, and function under an ecophysiological perspective. of the cellulose network. Other factors should be also taken into While no specific cuticle structural patterns could be identi- account, such as those affecting SE cuticles. fied in association with chemical composition, the background Together with cellulose, it is likely that other cuticular polysac- cell wall architecture seems to have an effect on cuticle morphol- charides, such as pectins or hemicelluloses, extend throughout ogy. We hence conclude that the existing methodologies do not the cuticle, as derived from cellulose and pectin distribu- fully enable to establish a direct link between cuticle composition tion in intact leaf cuticles of three model species (Guzmán and structure. Therefore, the implementation of new experimen- et al., 2014b). Under certain conditions, these polysaccharides tal approaches also analyzing the performance of pure chemical might form a gel-like phase with embedded cellulose fibrils, compounds (e.g., cutin monomers) may help us elucidate the as described for plant cell walls (Cosgrove, 2005; Burton et al., link between cuticle structure and chemistry also in relation with 2010). By analogy with the cell wall, the cellulose fibrils may different plant species, organs, and habitats. constitute the reinforcing rods of the cuticle, while the non- cellulosic phase may provide flexibility, mechanical support ACKNOWLEDGMENTS (Burton et al., 2010), and facilitate the transport of hydrophilic, The authors are grateful to Dr. Armando J. D. Silvestre (Univer- polar compounds and water through the cuticle (Guzmán et al., sity of Aveiro, Portugal) and Dr. M. Amparo Blázquez (University 2014a,b). of Valencia, Spain) for providing information about eucalypt

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soluble lipids and to Ramiro Martínez (Novozymes) for facili- Graça, J., and Lamosa, P. (2010). Linear and branched poly(ω-hydroxyacid) esters tating free enzyme samples. We also wish to thank M. Paz Andrés, in plant cutins. J. Agric. Food Chem. 58, 9666–9674. doi: 10.1021/jf1015297 Dr. Carlos Arrabal and Cristina Gutiérrez (Complutense Univer- Graça, J., Schreiber, L., Rodrigues, J., and Pereira, H. (2002). Glycerol and glyceryl esters of ω-hydroxyacids in cutins. Phytochemistry 61, 205–215. doi: sity of Madrid, Spain) for technical assistance with soluble lipid 10.1016/S0031-9422(02)00212-1 extraction and analysis. Thanks to Dr. J. A. Heredia-Guerrero for Guzmán, P., Fernández, V., Khayet, M., García, M. L., Fernández, A., and Gil, L. revising the FTIR spectroscopy results. Paula Guzmán is sup- (2014a). Ultrastructure of plant leaf cuticles in relation to sample preparation as ported by a pre-doctoral grant from the Technical University of observed by transmission electron microscopy. Scientific World J. 963921:9. doi: Madrid. Victoria Fernández is supported by a Ramón y Cajal 10.1155/2014/963921 Guzmán, P., Fernández, V., García, M. L., Khayet, M., Fernández, A., and Gil, L. contract (MINECO, Spain), co-financed by the European Social (2014b). Localization of polysaccharides in isolated and intact cuticles of eucalypt, Fund. poplar and pear leaves by enzyme-gold labelling. Plant Physiol. Biochem. 76, 1–6. doi: 10.1016/j.plaphy.2013.12.023 REFERENCES Guzmán, P., Gil, L., and Tadesse, W. (2013). Variation in growth traits and survival Baker, E. A. (1982). “Chemistry and morphology of plant epicuticular waxes,” of landraces of Eucalyptus globulus Labill in the Ethiopian highlands. 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(2014). in the absence of any commercial or financial relationships that could be construed The cuticle and plant defense to pathogens. Front. Plant Sci. 5:274. doi: as a potential conflict of interest. 10.3389/fpls.2014.00274 Silvestre, A. J. D., Cavaleiro, J. A. S., Delmond, B., Filliatre, C., and Bourgeois, G. Received: 02 July 2014; accepted: 30 August 2014; published online: 16 September 2014. (1997). Analysis of the variation of the essential oil composition of Eucalyptus Citation: Guzmán P, Fernández V, Graça J, Cabral V, Kayali N, Khayet M and Gil globulus Labill. from Portugal using multivariate statistical analysis. Ind. Crops L (2014) Chemical and structural analysis of Eucalyptus globulus and E. camal- Prod. 6, 27–33. doi: 10.1016/S0926-6690(96)00200-2 dulensis leaf cuticles: a lipidized cell wall region. Front. Plant Sci. 5:481. doi: Steinbauer, M. J., Davies, N. W., Gaertner, C., and Derridj, S. (2009). Epicuticu- 10.3389/fpls.2014.00481 lar waxes and plant primary metabolites on the surfaces of juvenile Eucalyptus This article was submitted to Plant Cell Biology, a section of the journal Frontiers in globulus and E. nitens (Myrtaceae) leaves. Aust. J. Bot. 57, 474–485. doi: Plant Science. 10.1071/BT09108 Copyright © 2014 Guzmán, Fernández, Graça, Cabral, Kayali, Khayet and Gil. This is Takada, S., and Iida, H. (2014). Specification of epidermal cell fate in plant shoots. an open-access article distributed under the terms of the Creative Commons Attribution Front. Plant Sci. 5:49. doi: 10.3389/fpls.2014.00049 License (CC BY). The use, distribution or reproduction in other forums is permitted, Tegelaar, E. W., Kerp, H., Visscher, H., Schenck, P. A., and de Leeuw, J. W. (1991). provided the original author(s) or licensor are credited and that the original publica- Bias of the paleobotanical record as a consequence of variations in the chemical tion in this journal is cited, in accordance with accepted academic practice. No use, composition of higher cuticles. Paleobiology 17, 133–144. distribution or reproduction is permitted which does not comply with these terms.

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